Alex Chenchick, York Y. Shu, Luda Diatchenko, Roger Li, Jason Hill and Paul D. Siebert. (Gene Cloning and Analysis Group, CLONETECH Laboratories, Pao Alto, CA, USA).

In: Gene Cloning and Analysis by RT-PCR. Edited by Paul Siebert and James Larrick. 1998

Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR.

Reaction mixture: 1 µM RTP; 1 µM TSO; 50–1000 ng total RNA; 2 mM DTT, 1 mM dNTP, 200 U SSII in 10 µL.

DNA/RNA ends tested: HO-G, Cap-G, HO-A, Cap-A, HO-C, Cap-C, HO-T

TSOs tested: rG, rGrG, rGrGrG, rGrGrGrGrG, rUrUrU, GGG, rGrGrG in all-r oligo.

With the wild-type MMLVm the HO-G DNA/RNA duplex is tailed with 1~5 extra nucleotides (Fig 2). Using radiolabelled nucleotides suggests that they are mostly Cs. "Not shown" experiments suggest that the presence of a cap "does not significantly influence the preference of addition of these non-templated nucleotides". The consensus tail is AACCC. SSII (RNAseH-) has a lower efficiency for adding nucleotides, compared with wild-type MMLV.

Template-switching is more efficient with at least 2 rG. dG is notably less efficient and rU has no visible efficiency (Figure 2).

In 2 % of the cDNAs, RT was primed by the TSO.